Cerebrospinal fluid control standard

ABSTRACT

A SYNTHETIC CONTROL STANDARD FOR THE DETERMINATION OF CERBROSPINAL FLUID CONSTITUENTS IS PROVIDED BY DILUTING NORMAL BLOOD SERUM WITH AN AQUEOUS SOLUTION OF GLUCOSE AND SALINE TO A TOTAL PROTEIN CONCENTRATION OF 30 TO 145 MG. PER 100 ML.

United States Patent "ce 3,753,925 CEREBROSPINAL FLUID CONTROL STANDARDAllan L. Louderback, Temple City, and Anthony J.

Fontana, Glendora, Calif., assignors to Baxter Laboratories, Inc.,Morton Grove, Ill. No Drawing. Filed Mar. 24, 1972, Ser. No. 237,905Int. Cl. G01n 31 /00 US. Cl. 252-408 3 Claims ABSTRACT OF THE DISCLOSUREA synthetic control standard for the determination of cerebrospinalfluid constituents is provided by diluting normal blood serum with anaqueous solution of glucose and saline to a total protein concentrationof 30 to 145 mg. per 100 ml.

This invention relates to a synthetic control standard for use in thedetermination of cerebrospinal fluid proteins and other constituents.

Cerebrospinal fluid is a clear, colorless fluid which is contained inthe subarachnoid spaces of the brain and spinal cord and the ventriclesof the brain. It contains a few lymphocytes and has a protein content ofabout 15 to 45 mg. per 100 ml. (mg. percent). Under certain patientconditions, for example, brain damage, brain or spinal tumors, it isdesired to assay the cerebrospinal fluid for total protein content andother parameters.

The normal adult total volume of cerebrospinal fluid is about 125 and150 ml. and it is renewed about every 3 to 4 hours. Normally, a spinalcord puncture will supply a specimen of only about 5 to ml. and repeatpunctures are usually to be avoided. In an average 6 ml. specimen, 4 ml.is generally used up in laboratory analysis for the patient from whichthe sample is obtained, leaving only about 2 ml. per patient forretention for other purposes. In a 300 bed hospital, it generally takeson the average about 9 months to collect one liter of surplus humancerebrospinal fluid. Thus, cerebrospinal fluids are precious specimens.

In the determination of cerebrospinal fluid protein, as with otherbiological fluid materials, it is desirable to employ control standardsof known composition for comparison with the patients sample.Heretofore, these control standards have comprised carefully collectedand processed human cerebrospinal fluid. However, because of therelatively small amount of material available, the product is costly.Moreover, because cerebrospinal (fluid is collected in such smallquantities, the material tends to deteriorate over the normal storageperiod required for collection of a suitable quantity for sale such as aliter or more. Enzymes in the fluid will tend to react with otherconstituents and product break-down products. Because of the numeroussmall samples which make up any collection of significant volume, therisk of bacterial contamination is greater than in ordinary blood serumcollection. Furthermore, the human cerebrospinal fluid material isdiflicult to handle in processing since it does not lyophilize readily.Because its mass is relatively small, it forms a fine powder which tendsto collect on the upper walls and closure of the sample bottles with aconsequent loss of material upon opening the closure and reconstitutionwith water prior to use.

Accordingly, it is an object of this invention to provide a syntheticcontrol standard for use in the determination of cerebrospinal fluid.

Patented Aug. 21, 1973 It is another object of this invention to providea synthetic cerebrospinal fluid control standard which has improvedlyophilization handling characteristics over the naturally occurringmaterial. Other objects and advantages of the invention will be apparentto those skilled in the art upon reading of the disclosure hereof.

In accordance with the invention, a synthetic control standard for thedetermination of cerebrospinal fluid is prepared from normal blood serumby dilution with a reagent comprising glucose and chloride ion inaqueous solution to a level whereby the total protein concentrationranges from about 30 to about 145 mg. percent. It is preferred that thediluted serum contains from about 40 to about 200 mg. percent glucoseand from about to about 130 meq. per liter of chloride ion.

The chloride ion can be provided by use of an aqueous solution of analkali metal chloride, preferably sodium chloride. The chloride salt andthe glucose preferably are analytical or reagent grade materials.

For some purposes it is desired to include in the synthetic controlstandard of this invention a small amount of pre-albumin, preferablyfrom about 1 to about 3 mg. percent. Pre-alburnin is a special fractionobtained from blood serum and is described, for example, by Got, et al.,Isolement et caractrisation dune pralbumine du serum humain, Protides ofthe Biological Fluids, Vol. 10, H. Peeters, ed., Amsterdam, Elsevier,1963, pp. 125-126; Schultze and Hermans, Molecular Biology of HumanProtein, Vol. 1, Amsterdam, Elsevier, 1965, pp. l84-l85.

After dilution of the blood serum with aqueous glucose and saline orother chloride containing ion, with or without the added pre-albumin,the aqueous solution is lyophilized to a dry material. The lyophilizedmaterial is improved by the addition of a small amount of gum acaciaprior to lyophilization, for example, on the order of about 0.1%. Thisimproved lyophilized, dry material can be readily reconstituted withwater prior to use without product loss on the container closure.

The synthetic cerebrospinal fluid control standard made as describedherein has been found to produce much sharper bands in theelectrophoretic pattern in actual use than obtained with the normalhuman cerebrospinal fluid. This is an added advantage of the inventionin practice and enables the technician to improve the accuracy of thecontrol procedures.

The following examples will further illustrate the invention, althoughthe invention is not limited to these specific examples.

EXAMPLE 1 A synthetic control standard for use in the determination ofcerebrospinal fluid is prepared as follows:

Human blood serum from which the clotting factors have been removed isdiluted with an aqueous solution of glucose and sodium chloridecontaining 6.38 grams of NaCl and 600 mg. of glucose per liter. Theserum is thereby diluted to a total protein concentration of 110 mg. perml. by admixing 65 parts by volume of the diluent solution with one partby volume of the serum. To one liter of the diluted serum is then added10 ml. of a 10% solution of gum acacia in water. The resulting solutionis then filled in 3 ml. aliquots into 5 ml. bottles and lyophilized. Thelyophilized, dry material is stored at 2 to 8 C. until required for use.In use, the dry material is reconstituted by dilution with 3 ml. ofdistilled water per bottle. Upon reconstitution of the foregoingmaterial, the following assay values were determined.

Constituent: Value Chloride, meq. per liter 105 Glucose, mg. per 100 ml.57 Protein, total, mg. per 100 ml. 110 Pre-albumin, percent of totalAlbumin, percent of total 58 a -globulin, percent of total 6 a-globulin, percent of total 12 fl-globulin, percent of total l3-globulin, percent of total 11 Colloidal gold (Langes test) 1111100000EXAMPLE 2 Example 1, above, is repeated except that 1-3 mg. percent ofpre-albumin is added to the solution prior to lyophilization.

The control standards prepared in Examples 1 and 2, above, are used inthe same manner as an unknown cerebrospinal fluid for comparison withthe patients sample to provide a check on the analytical determination.

Various other examples and modifications of the foregoing examples canbe made by the person skilled in the art after reading the foregoingdescription and the appended claims without departing from the spiritscope of the invention. All such further examples and modifications areincluded Within the scope of the appended claims.

What is claimed is:

1. The method of making a synthetic control standard for use in thedetermination of cerebrospinal fluid constituents comprising dilutingnormal blood serum with an aqueous diluent containing from about toabout 200 mg. per 100 ml. of glucose and from about to about 130milliequivalents per liter of chloride ion to a level at which the totalprotein concentration ranges from about 30 to about 145 mg. per ml.

2. The method of claim 1 including the additional steps of admixing theliquid control standard with about 0.1% gum acacia and lyophilizing to adry, powdered material.

3. A synthetic control standard for use in the determination ofcerebrospinal fluid comprising normal blood serum diluted with anaqueous diluent containing from about 40 to about 200 mg. per 100 ml. ofglucose and from about 80 to about milliequivalents per liter ofchloride ion to a level at which the total protein concentration rangesfrom about 30 to about mg. per 100 ml.

References Cited UNITED STATES PATENTS 3,627,468 12/1971 Goldenberg etal. 2323() B MAYER WEINBLA'IT, Primary Examiner U.S. Cl. X.R. 23-23O B;424-2

